The research proposed in this competing renewal application will continue and extend investigations on the utilization of selected aromatase inhibitors as biochemical tools in studying the active site of aromatase. Investigations by the P.I. have demonstrated enhanced affinity of microsomal aromatase for several 7alpha-thioether-substituted 4-androstene- 3,17-diones and 1,4-androstadiene-3,17-diones. New 7-sub-stituted C19- steroids with enhanced affinity for the enzyme and/or greater metabolic stability have been developed and used in structure-activity studies of aromatase inhibition. The overall goal of our research is to use specific aromatase inhibitors to map the steroid binding site and study the structure of the aromatase cytochrome P450 protein. Investigations of the interactions of these 7-substituted steroidal inhibitors with the aromatase enzyme complex have recently focused on employing purified cytochrome P450arom. Isolation and purification of cytochrome P450arom from human placental microsomes is accomplished employing sodium cholate solubilization, ammonium sulfate fractionation, and gel chromatography. Two radiolabeled enzyme-activated inhibitors are being examined, and biochemical studies recently demonstrated direct evidence of covalent binding of these inhibitors to cytochrome P450arom. The specific aims of this renewal application are: (1) Further examination of the extent of covalent binding of radiolabeled enzyme-activated inhibitors with purified aromatase preparations will be performed. The stoichiometry of the covalent binding will be determined for the radiolabeled inhibitors. (2) Inhibitor-bound aromatase protein will be isolated and treated with proteolytic enzymes, and peptides will be separated by HPLC. The amino acid sequence of radiolabeled peptide fragments will be determined by microsequence analysis. (3) New radiolabeled [14C]- and [3H]- analogs of selected enzyme-activated or affinity inhibitors will be prepared. These inhibitors will be utilized in the examination of various regions in the steroid binding site. Also, certain radiolabeled inhibitors will be employed to study the mechanism of the enzyme-activated irreversible binding. (4) Molecular modeling and computational chemistry will be utilized in determination of possible conformations of the active site of cytochrome P450arom and possible interactions with inhibitors. Thus, radiolabeled analogs of selected inhibitors will be prepared and utilized to probe the active site of purified aromatase and the mechanism of inactivation. Knowledge of the chemistry of the enzymatic "pocket" of aromatase is critical in the further design and development of more effective and potent agents for the control of estrogenic processes and for the treatment of advanced estrogen-dependent breast cancer.